The invention relates generally to diagnostic methods for real time diagnosis of biological cells and tissue. A diagnosis of abnormal tissue can be made by the detection of differences in properties of biological cells, properties such as cell density, size and composition. A diagnosis of abnormal tissue may also include a characterization of these differences in cellular properties. In addition to aiding a health care provider with making a diagnosis of abnormal tissue, an apparatus for diagnosis that provides real-time imaging ensures that the abnormal tissues is also completely removed during a surgical procedure so that the subject does not have to undergo multiple surgical procedures to remove all traces of the abnormal tissue. Typically it takes 2 to 5 days to obtain a conclusive answer on the surgical success which is determined after detailed pathology and histology analysis is performed on the sample. Real-time detection would give feedback to the surgeon during the surgery and thereby reducing the possibility that the subject will have to undergo a 2nd surgery due to the presence of “positive margins”, or not enough cancer-free margins on the excised tissue.
Current devices and methods for detecting abnormal tissue in a sample have several disadvantages. The methods currently used, include X-rays, ultrasound imaging, magnetic resonance imaging, thermal imaging, radiofrequency (RF) reflection and absorption, and electrical impedance techniques. Electrical impedance techniques has the disadvantage that the detection of abnormalities in the tissues and the cells is done by measuring the changes in electrical impedance of the tissue globally rather than measuring electrical impedance locally because the current devices and methods are positioned outside of the body when in use. Apparatuses that use global measures to detect changes in electrical impedance are less sensitive and have relatively poor spatial resolution. For example, in X-ray imaging the sensitivity of the device in imaging small-size cancer lumps such as lumps that are less than 3 mm in size is low. Additionally, in cases where there is a low relative amount of malignant cells adjacent to benign cells, the sensitivity of the X-ray is less than 30%. X-rays are also affected by any other objects that may absorb the X-rays, such as a dense tissue structures or bone located between the X-ray source and the detector. Additionally, an X-rays machine cannot be used inside the body.
Another technique for detecting the presence of abnormal tissue is the use of ultrasound waves to detect cancer cells. Ultrasound machines image tissue by looking at the reflection of the ultrasound waves of the denser cells. Also the elasticity differences between benign and malignant cells contribute to the image produced by ultrasound. The use of ultrasound is further limited in the minimum size of detectable abnormal tissue because ultrasound imaging of smaller sizes is subject to poorer signal to noise ratio. Similarly, it is also difficult to detect changes in cell density of the tissue in denser media.
Another imaging apparatus used to detect the presence of abnormal tissue is the MRI machine. Like the other imaging apparatuses previously mentioned, MRI images are also affected by cell density and composition. Further, the MRI image is strongly affected by the amount of background noise from the overall tissue scanned and is also limited by the size of abnormal tissue that is detectable. And like X-ray, MRI cannot be used inside of the body.
Other imaging methods include: thermal imaging techniques which detect changes in the temperature of tissues that have denser cell densities and which attract more blood flow to the area; RF reflection and absorption, which is also used to detect cancer cells by detecting variations in the reflection and absorption of RF in suspect tissues as compared to benign cells; electrical impedance techniques have also been developed to determine the malignancy of the cells within the organ by monitoring electrical responses from the outer surface of the tissue.
Sometimes a tissue requires further analysis. For example, it may be beneficial to know whether a sample of abnormal cells is benign or malignant. It may, therefore, be necessary to send the sample out for analysis to a pathology lab. In the pathology lab, cancerous cells are characterized using histological methods which can be time consuming and may involve complex sample preparation procedures that can last anywhere from 8-12 hours.
Because the current techniques mentioned above are typically capable only of being positioned outside of the body there may be difficulty in detecting small volumes of abnormal tissue or testing small sample areas. Also, positioning the detecting device outside of the body creates the potential for a greater amount of interference with neighboring tissue; makes it more difficult to reach the target tissue through structures in between the target tissue and the testing device; and increase the likelihood that the signal to noise ratio will be poor. In addition, the current methods for detecting abnormal tissue employ bulky machinery. Further, tissue samples are currently sent to pathology labs for testing which ultimately increases the time frame for making a diagnosis.
A method currently being used for detecting and identifying variations in samples is the optical detection of cellular variations. Optical techniques for diagnosing include the use of Surface Enhanced Raman Spectroscopy (SERS) as applied to tissues. Raman spectroscopy has been shown to have very good sensitivity down to the single cell level. In SERS electromagnetic radiation is introduced to a sample through an optical probe. Treating the surface of the structure for holding the sample with electrically conductive, metallic materials enhances the electromagnetic field experienced by the sample. The electromagnetic radiation interacts with the sample and is scattered and reflected back to a detector due to inherent properties of the sample. The scattered electromagnetic radiation can be used as an indicator of the characteristics of the tissue sample. This technique has been shown to have good sensitivity and specificity for cancer cells. However, the technique works on thin tissue sections and requires the use of a microscopy set-up.
Thus, there exists a need for optical devices and methods that detect in real-time the near-field effects of electromagnetic radiation on abnormal tissue with high sensitivity and precision and which is capable of being contained in a compact unit that is easily manipulated in reference to the sample. In addition to the advantage that the invention described herein provides real-time diagnosis of the sample being tested, an automated real-time diagnosis instrument will help to eliminate the possibility of human error or missing a critical volume of tissue.